Direct Readout of Multivalent Chromatin Reader-Nucleosome Interactions by Nucleosome Mass Spectrometry.

Histone post-translational modifications (PTMs) often serve as distinct recognition sites for the recruitment of chromatin-associated proteins (CAPs) for epigenome regulation. While CAP-PTM interactions have been extensively studied using histone peptides, this cannot consider the regulatory potential of multi-site binding on intact nucleosomes. To overcome this limitation, we applied Nucleosome Mass Spectrometry (Nuc-MS), a native Top-Down MS approach that enables controlled disassembly of intact CAP:nucleosome (CAP:nuc) complexes to provide a direct readout of the contained histone proteoforms.

G34R cancer mutation alters the conformational ensemble and dynamics of the histone H3.3 tails.

In recent years, mutations in the histone variant H3.3 have been discovered in pediatric and adult gliomas and osteosarcomas. One of these is the G34R mutation in the H3.

Multiple transactivation domains of EZH2 bind to the TAZ2 domain of p300 and stimulate the acetyltransferase function of p300.

The H3K27me-specific methyltransferase enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of the repressive complex Polycomb repressive complex 2. EZH2 is typically implicated in transcriptional silencing, but it can also activate gene expression. Here, we show that EZH2 contains three adjacent transactivation domains (EZH2TAD) that are recognized by the TAZ2 domain of the transcriptional coactivator and acetyltransferase p300 (p300TAZ2).

Polyamines regulate cell fate by altering the accessibility of histone tails.

Polyamines are polycationic alkyl-amines abundant in proliferating stem and cancer cells. How these metabolites influence numerous cellular processes remains unclear. Here we show that polyamine levels decrease during differentiation and that inhibiting polyamine synthesis leads to a differentiated-like cell state.

Nucleosome conformation dictates the histone code.

Histone post-translational modifications (PTMs) play a critical role in chromatin regulation. It has been proposed that these PTMs form localized 'codes' that are read by specialized regions (reader domains) in chromatin-associated proteins (CAPs) to regulate downstream function. Substantial effort has been made to define [CAP: histone PTM] specificities, and thus decipher the histone code and guide epigenetic therapies.

Protocol to prepare doubly labeled fluorescent nucleosomes for single-molecule fluorescence microscopy.

Single-molecule fluorescence microscopy (SMFM) has been shown to be informative in understanding the interaction of chromatin-associated factors with nucleosomes, the basic building unit of chromatin. Here, we present a protocol for preparing doubly labeled fluorescent nucleosomes for SMFM. We describe steps for over-expression in E.

PBRM1 bromodomains associate with RNA to facilitate chromatin association.

PBRM1 is a subunit of the PBAF chromatin remodeling complex, which is mutated in 40-50% of clear cell renal cell carcinoma patients. It is thought to largely function as a chromatin binding subunit of the PBAF complex, but the molecular mechanism underlying this activity is not fully known. PBRM1 contains six tandem bromodomains which are known to cooperate in binding of nucleosomes acetylated at histone H3 lysine 14 (H3K14ac).

Single-Molecule Characterization of Cy3.5 -Cy5.5 Dye Pair for FRET Studies of Nucleic Acids and Nucleosomes.

Single molecule FRET (Forster resonance energy transfer) is very powerful method for studying biomolecular binding dynamics and conformational transitions. Only a few donor - acceptor dye pairs have been characterized for use in single-molecule FRET (smFRET) studies. Hence, introducing and characterizing additional FRET dye pairs is important in order to widen the scope of applications of single-molecule FRET in biomolecular studies.

Visualizing Conformational Ensembles of the Nucleosome by NMR.

The formation of chromatin not only compacts the eukaryotic genome into the nucleus but also provides a mechanism for the regulation of all DNA templated processes. Spatial and temporal modulation of the chromatin structure is critical in such regulation and involves fine-tuned functioning of the basic subunit of chromatin, the nucleosome. It has become apparent that the nucleosome is an inherently dynamic system, but characterization of these dynamics at the atomic level has remained challenging.

Reprogramming CBX8-PRC1 function with a positive allosteric modulator.

Canonical targeting of Polycomb repressive complex 1 (PRC1) to repress developmental genes is mediated by cell-type-specific, paralogous chromobox (CBX) proteins (CBX2, 4, 6, 7, and 8). Based on their central role in silencing and their dysregulation associated with human disease including cancer, CBX proteins are attractive targets for small-molecule chemical probe development. Here, we have used a quantitative and target-specific cellular assay to discover a potent positive allosteric modulator (PAM) of CBX8.

Nucleosome composition regulates the histone H3 tail conformational ensemble and accessibility.

Hexasomes and tetrasomes are intermediates in nucleosome assembly and disassembly. Their formation is promoted by histone chaperones, ATP-dependent remodelers, and RNA polymerase II. In addition, hexasomes are maintained in transcribed genes and could be an important regulatory factor.

Characterization of functional disordered regions within chromatin-associated proteins.

Intrinsically disordered regions (IDRs) are abundant and play important roles in the function of chromatin-associated proteins (CAPs). These regions are often found at the N- and C-termini of CAPs and between structured domains, where they can act as more than just linkers, directly contributing to function. IDRs have been shown to contribute to substrate binding, act as auto-regulatory regions, and drive liquid-liquid droplet formation.

Histone Tail Conformations: A Fuzzy Affair with DNA.

The core histone tails are critical in chromatin structure and signaling. Studies over the past several decades have provided a wealth of information on the histone tails and their interaction with chromatin factors. However, the conformation of the histone tails in a chromatin relevant context has remained elusive.

Evolutionary Conservation of Structural and Functional Coupling between the BRM AT-Hook and Bromodomain.

The BAF chromatin remodeling complex is critical for genome regulation. The central ATPase of BAF is either BRM or BRG1, both of which contain a C-terminal bromodomain, known to associate with acetylated lysines. We have recently demonstrated that in addition to acetyl-lysine binding, the BRG1/BRM bromodomain can associate with DNA through a lysine/arginine rich patch that is adjacent to the acetyl-lysine binding pocket.

The molecular basis of selective DNA binding by the BRG1 AT-hook and bromodomain.

The ATP-dependent BAF chromatin remodeling complex plays a critical role in gene regulation by modulating chromatin architecture, and is frequently mutated in cancer. Indeed, subunits of the BAF complex are found to be mutated in >20% of human tumors. The mechanism by which BAF properly navigates chromatin is not fully understood, but is thought to involve a multivalent network of histone and DNA contacts.

Optimization of Ligands Using Focused DNA-Encoded Libraries To Develop a Selective, Cell-Permeable CBX8 Chromodomain Inhibitor.

Polycomb repressive complex 1 (PRC1) is critical for mediating gene expression during development. Five chromobox (CBX) homolog proteins, CBX2, CBX4, CBX6, CBX7, and CBX8, are incorporated into PRC1 complexes, where they mediate targeting to trimethylated lysine 27 of histone H3 (H3K27me3) the N-terminal chromodomain (ChD). Individual CBX paralogs have been implicated as drug targets in cancer; however, high similarities in sequence and structure among the CBX ChDs provide a major obstacle in developing selective CBX ChD inhibitors.

The PHD finger of Spp1 mediates histone modification cross-talk.

Binding of the Spp1 PHD finger to histone H3K4me3 is sensitive to adjacent post-translational modifications in the histone tail. This commentary discusses the findings of He and colleagues [, 1957-1973] which show that the PHD finger binds to H3K4me3 in a selective manner which is conserved in the and mammalian orthologues of Spp1.

RNA Splicing of the BHC80 Gene Contributes to Neuroendocrine Prostate Cancer Progression.

Background: Prostate adenocarcinoma (AdPC) progression to treatment-induced neuroendocrine prostate cancer (t-NEPC) is associated with poor patient survival. While AdPC and t-NEPC share similar genomes, they possess distinct transcriptomes, suggesting that RNA splicing and epigenetic mechanisms may regulate t-NEPC development.

Objective: To characterize the role of alternative RNA splicing of the histone demethylase BHC80 during t-NEPC progression.

Strategies for Generating Modified Nucleosomes: Applications within Structural Biology Studies.

Post-translational modifications on histone proteins play critical roles in the regulation of chromatin structure and all DNA-templated processes. Accumulating evidence suggests that these covalent modifications can directly alter chromatin structure, or they can modulate activities of chromatin-modifying and -remodeling factors. Studying these modifications in the context of the nucleosome, the basic subunit of chromatin, is thus of great interest; however, the generation of specifically modified nucleosomes remains challenging.

The EZH2 SANT1 domain is a histone reader providing sensitivity to the modification state of the H4 tail.

SANT domains are found in a number of chromatin regulators. They contain approximately 50 amino acids and have high similarity to the DNA binding domain of Myb related proteins. Though some SANT domains associate with DNA others have been found to bind unmodified histone tails.

Engagement of DNA and H3K27me3 by the CBX8 chromodomain drives chromatin association.

Polycomb repressive complex 1 (PRC1) is critical for mediating gene repression during development and adult stem cell maintenance. Five CBX proteins, CBX2,4,6,7,8, form mutually exclusive PRC1 complexes and are thought to play a role in the association of PRC1 with chromatin. Specifically, the N-terminal chromodomain (CD) in the CBX proteins is thought to mediate specific targeting to methylated histones.

E-C coupling structural protein junctophilin-2 encodes a stress-adaptive transcription regulator.

Junctophilin-2 (JP2) is a structural protein required for normal excitation-contraction (E-C) coupling. After cardiac stress, JP2 is cleaved by the calcium ion-dependent protease calpain, which disrupts the E-C coupling ultrastructural machinery and drives heart failure progression. We found that stress-induced proteolysis of JP2 liberates an N-terminal fragment (JP2NT) that translocates to the nucleus, binds to genomic DNA, and controls expression of a spectrum of genes in cardiomyocytes.